Some probiotic Lactobacillus strains demonstrated intrinsic adjuvanticity and enhanced antigen-specific immune responses to rotavirus infection and to viral and bacterial vaccines. However, the underlying mechanism of the adjuvant effect is undefined. We hypothesize that these Lactobacillus strains, rich in CpG motifs in the genome, enhance vaccine immunogenicity via immunostimulatory effects on antigen-presenting cells (APCs), which in turn, promote type 1 immune responses. We will use a well-characterized gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea to identify the mechanisms of lactobacilli's immunostimulating effects through studies of innate and adaptive immune responses and their regulation. Our specific aims are to: 1. Determine the immunogenicity and protective efficacy of the live oral attenuated HRV (AttHRV) vaccine with Lactobacillus acidophilus and L. rhamnosus GG as adjuvants in Gn pigs and identify the mechanism of the adjuvant effects. The efficacy of lactobacilli-adjuvanted AttHRV vaccines against rotavirus diarrhea in Gn pigs challenged with a virulent HRV will be evaluated. The mechanism of immunostimulation will be identified by determining the effect of probiotics on dendritic cell (DC) maturation and function, toll-like receptor (TLR)9-expressing APC responses, Th1, Th2, and T regulatory cell and corresponding cytokine responses, and effector and memory B cell responses in intestinal and systemic sites of Gn pigs. A subset of experiments will be performed in an immunoglobulin heavy chain gene knockout (Ig-KO) and CD8+ T cell depleted Gn pig challenge model to clearly identify determinants of protective immunity against rotavirus diarrhea. 2. Evaluate the effects of lactobacilli on the immunogenicity and protective efficacy of the AttHRV vaccine in "conventionalized" Gn pigs. Use of conventionalized Gn pigs (mimicking formula-fed human infants colonized with commensal microflora) facilitates extrapolating our findings to humans and prevents interference and confounding factors from wild swine rotavirus contaminations. The innovative aspects of this research application include (1) use of probiotics as adjuvants rather than direct modulators of disease;and (2) use of Gn pigs, Ig-KO Gn pigs, and conventionalized Gn pigs to study the efficacy and mechanisms of probiotics'adjuvant effect on rotavirus vaccine and the determinant of protective immunity. Knowledge generated will provide a foundation for the clinical uses of probiotics as adjuvants to enhance protective efficacies of vaccines against rotavirus and other enteric virus induced diseases. Our long term goal is to promote clinical trials using the lactobacilli as rotavirus vaccine adjuvant. Considering the heavy disease burden and huge economic losses due to rotavirus infection worldwide, any improvement in vaccine efficacies will have a major impact on improving human health. Public Health Relevance: Probiotic Lactobacillus acidophilus and L. rhamnosus GG have shown intrinsic adjuvanticity and enhanced virus-specific immune responses to rotavirus natural infections and live oral vaccines;however, mechanisms for the adjuvanticity of probiotics have not been clearly defined. Our study will identify the underlying mechanisms by which probiotics exert the adjuvant effects and this knowledge will facilitate the clinical uses of probiotic adjuvants for vaccines against rotavirus and other enteric virus-induced diseases. Rotavirus gastroenteritis poses a tremendous ongoing disease burden in the US and worldwide, with costs to health care and society exceeding one billion dollars annually in the US and even more immense morbidity and mortality in developing countries;thus, any improvement in vaccine efficacies will have a major impact on improving human health.